Project Summary/Abstract Despite recent advances in cancer treatment, pancreatic cancer (PDA) remains a devastating disease with increasing incidence. PDA is highly refractory to the current standards of care which is due, in part, to a rewired metabolism that supports growth in an austere tumor microenvironment. Therefore, elucidating the metabolic pathways critical for tumor growth is imperative to understanding the complexities of PDA biology as well as identifying novel therapeutic opportunities. There is emerging evidence, however, that in vitro metabolism may not be entirely reflective of the metabolic requirements of a tumor. Due to the difficulties of studying metabolism in vivo with conventional techniques, a functional genetic CRISPR screening approach was employed to determine the metabolic liabilities of PDA tumors and comprehensively define critical metabolic pathways for primary tumor growth and metastasis. In the F99 phase of the proposal, I analyzed CRISPR screening data to create a map of PDA metabolic dependencies in vivo. I will now use this map of PDA metabolic dependencies to determine critical pathways to support tumor growth and evaluate these pathways as potential therapeutic targets in orthotopic mouse models of pancreatic cancer. I will also determine the mechanism by which a metabolic pathway that supports growth in vivo is not required in vitro. Preliminary analysis of the CRISPR screen suggest that heme biosynthesis is required in vivo but is not essential to growth of PDA cells in vitro. I have designed a series of experiments to determine how heme biosynthesis supports growth in tumors and how this dependency is masked by in vitro culture conditions. In the K00 phase of this proposal, I will shift focus from the metabolism primary tumor to metastasis and the immune system. PDA is highly metastatic and metastasis is the primary cause of mortality for most cancer patients. Understanding the differences between the metabolism of primary and metastatic cancer will offer insight into how to treat metastasized tumors and how to target cancer cell metabolism to increase immune surveillance of circulating tumor cells. The proposed work will be the first comprehensive analysis of pathways that support PDA growth in vivo and will help identify candidates for therapeutic intervention. These experiments will also determine the metabolic differences between primary and metastatic tumors and how cancer cell metabolism interacts with the host?s immune system. These data will inform what metabolic pathways lead to immune evasion and if targeting these pathways therapeutically will lead to a reduced primary tumor growth and metastatic tumor burden.